With the use of the photoacoustic spectrometry system, in which a mixture of lipid- and water-soluble dyes is applied to the skin and then irradiated with light from a xenon lamp (425 nm and 550 nm), we measured photoacoustic signals of both dyes within the stratum corneum and their disappearance rate through the stratum corneum. The signal intensity was higher and dyes penetrated faster in clinically normal skin of patients with atopic dermatitis (AD) compared with healthy subjects, indicating an impairment of the in vivo cutaneous permeability barrier function against both lipophilic and hydrophilic chemicals. Furthermore, penetration rates of the hydrophilic dyes tended to increase in proportion to the severity of AD and significantly correlated with serum IgE levels in the severe AD group. Thus, abnormal barrier functions of clinically normal skin in AD may predispose inflammatory processes evoked by irritants and allergens, especially their water-soluble elements.